Functionalised thermally induced phase separation (TIPS) microparticles enabled for “click” chemistry† †Electronic supplementary information (ESI) available. See DOI: 10.1039/d0ob00106f

In this study we describe a novel platform for generating functionalised TIPS microparticles for “click” conjugation to various active compounds.

buffer, heated for 3 minutes at 75 °C and loaded onto the gel with a total volume of 5 µL.
Samples were run at constant current (30 mA) for 40 minutes in 1 x SDS running buffer and stained with Coomasie.

Protein LCMS
All proteins were prepared for analysis by repeated diafiltration into ammonium acetate buffer (50 mM ammonium acetate, pH 6.9) using VivaSpin sample concentrators (GE Healthcare, 10000 MWCO) to a concentration of 2 µM. Samples were submitted to the UCL Chemistry Mass Spectrometry Facility at the Chemistry Department, UCL for analysis on the Agilent 6510 QTOF LC-MS system (Agilent, UK). 10 µL of each sample was injected onto a PLRP-S, 1000A, 8 mM, 150 mm x 2.1 mm column, which was maintained at 60 °C. Flow rate was set at 0.600 ml/min. Solvent A is H2O (0.1% formic acid), solvent B is MeCN (0.1% formic acid), and separation was achieved using a gradient elution. The column effluent was continuously electrosprayed into the capillary ESI source of the Agilent 6510 QTOF mass spectrometer and ESI mass spectra were acquired in positive electrospray ionisation (ESI) mode using the m/z range 1000 -8000 in profile mode. The raw data was converted to zero charge mass spectra using maximum entropy deconvolution algorithms using MassHunter software (version B.07.00).

Fabrication of TIPS Microparticles
TIPS microparticles intended for click chemistry were prepared by blending together two polymers: poly(D, L-lactide-co-glycolide) (PLGA; PURASORB PDLG 7507 75/25 DLlactide/glycolide copolymer; inherent viscosity 0.75 dl/g; Corbion, Netherlands) and PLGA azide-capped (PLGA-N3; Mw ~95K; Nanosoft Polymers, USA). Each polymer was dissolved separately in dimethyl carbonate (Sigma Aldrich, Dorset, UK) using magnetic stirring to produce a 5 wt% polymer solution before specific volumes were mixed together to produce solutions with different v/v ratios of PLGA: 25:75,50:50,75:25,100:0). The polymer solutions were mixed together by placing on a vortex for 10 seconds. The polymer solution was immediately fed into a Nisco Encapsulator Unit (Nisco Engineering, Zurich, Switzerland; Frequency: 2.75 kHz, Amplitude: 70%) by a syringe pump (Harvard Apparatus, Kent, UK), at a constant flow rate of 2 mL/min. The polymer droplets were formed using a 150 µm sapphire nozzle and collected in a liquid nitrogen quenching bath. Frozen solvent was removed from the polymer droplets by lyophilisation for 48 h. The dried TIPS microparticles were stored in a desiccator at room temperature in rubber stoppered glass vials under vacuum.

Imaging Click-TIPS microparticles
Fluorescence microscopy was used to detect azide-capped PLGA on the surface of TIPS microparticles via staining with an azadibenzocyclooctyne-tetramethyl rhodamine between different sample groups the auto exposure was disabled and imaging parameters were set using 0% azide (100% PLGA) microparticle before acquiring all images using the same camera settings.
Scanning electron microscopy was used to assess the size distribution of the microparticles and the surface ultrastructural features of TIPS microparticles composed of the different ratios of PURASORB PDLG 7507 and azide-capped PLGA. The microparticles were placed onto aluminium stubs and coated with gold for 60 seconds using a Quorum Technologies Q150R ES gold coater. The samples were imaged using a Hitachi S3400N scanning electron microscope. The diameter of 60 microparticles from the SEM images was measured using ImageJ software.

Anti-Fab HRP modified ELISA
Microparticles ( 100 µL of supernatant was transferred to a 96-well plate and the absorbance was read at 490 nm.